The majority of early-onset familial Alzheimer's disease (FAD) has recently been demonstrated to be caused by mutations in two novel genes termed presenilin 1 (PS1) and presenilin 2 (PS2). Although the normal biological role(s) of these two proteins and the details of the mechanism by which the FAD-associated mutations exert their effect remain unknown, it has recently been demonstrated that fibroblasts from affected individuals harboring certain of these mutations have increased levels of Abeta42. To date, little is known about the basic biological characteristics of the presenilins. Clearly future studies must be aimed at determining the normal roles of these two proteins and how they are linked to the amyloid precursor protein (APP) and the production of Abeta. In the current proposal we have elected to focus on one member of the presenilin family, PS2. We propose to perform a series of experiments which are aimed at providing rudimentary but critical information about PS2 and the effects that FAD-associated mutation(s) have on its fundamental properties. In the first two aims we will expand upon our preliminary findings and continue the analysis of the expression, processing, and subcellular localization of PS2, while concurrently examining the effects of the N141I mutation on these characteristics. Because of the apparent association between the presenilin mutations and the generation of Abeta, the goals of the second two aims are to determine whether there is a detectable interaction between PS2 and APP, and to examine whether quantitative or qualitative changes in PS2 have an effect on the processing of APP. Our overall goal is to provide first order information for studies aimed at determining both the normal biological role of PS2 and the mechanism by which mutations in this protein lead to the neurodegeneration and synaptic loss in early-onset Alzheimer's disease. The results of the proposed experiments should help to not only elucidate the role of PS2 in the pathophysiology of Alzheimer's disease, but also to provide a solid framework on which to ultimately base effective therapeutic strategies and future experiments. Specifically, we plan to: l) Continue our analysis of the effects that mutations have on the processing & localization of PS2 itself; 2) Define which domains of PS2 dictate its intracellular localization to the Golgi/ER, and assess whether particular regions of PS2 that are not conserved in PS1 cause the intracellular localization pattern of PS2 to be slightly different from that of PS1; 3) Determine whether the N141l PS2 mutation or changes in the level of PS2 alter the processing or subcellular localization of APP; 4) Assess whether there is a detectable interaction between APP and PS2 and, if so whether the N141I mutation alters this interaction. Although we will first concentrate on analysis of the N141I mutation, we will also assess the effects of other PS2 mutations as they are described.